Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model

Oxygen starvation triggers the shiftdown of the obligate aerobe Mycobacterium bovis BCG to a state of dormancy. Two-dimensional electrophoresis showed a drastic up-regulation of the alpha-crystallin homolog, the putative response regulator Rv3133c, and the two conserved hypothetical proteins Rv2623 and Rv2626c in dormant bacilli.     

Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.     

Hematopoietic cell fate and the initiation of leukemic properties in primitive primary human cells are influenced by Ras activity and farnesyltransferase inhibition

The Ras pathway transduces divergent signals determining normal cell fate and is frequently activated in hematopoietic malignancies, but the manner in which activation contributes to human leukemia is poorly understood. We report that a high level of activated H-Ras signaling in transduced primary human hematopoietic progenitors reduced their proliferation and enhanced monocyte/macrophage differentiation. However, the exposure of these cells to a farnesyltransferase inhibitor and establishment of a moderate level of Ras activity showed increased proliferation, an elevated frequency of primitive blast-like cells, and progenitors with enhanced self-renewal capacity. These results suggest that the amplitude of Ras pathway signaling is a determinant of myeloid cell fate and that moderate Ras activation in primitive hematopoietic cells can be an early event in leukemogenesis.     

Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.     

Lipoprotein kinetics in the metabolic syndrome: pathophysiological and therapeutic lessons from stable isotope studies

Dyslipoproteinaemia is a cardinal feature of the metabolic syndrome that accelerates atherosclerosis. It is usually characterised by high plasma concentrations of triglyceride-rich and apolipoprotein (apo) B-containing lipoproteins, with depressed concentrations of high-density lipoprotein (HDL). Dysregulation of lipoprotein metabolism in these subjects may be due to a combination of overproduction of very-low-density lipoprotein (VLDL) apoB-100, decreased catabolism of apoB-containing particles, and increased catabolism of HDL apoA-I particles. These abnormalities may be consequent on a global metabolic effect of insulin resistance that increases the flux of fatty acids from adipose tissue to the liver, the accumulation of fat in the liver, the increased hepatic secretion of VLDL-triglycerides and the remodelling of both low-density lipoprotein (LDL) and HDL particles in the circulation; perturbations in lipolytic enzymes and lipid transfer proteins contribute to the dyslipidaemia. Our in vivo understanding of the kinetic defects in lipoprotein metabolism in the metabolic syndrome has been chiefly achieved by ongoing developments in the use of stable isotope tracers and mathematical modelling. Knowledge of the pathophysiology of lipoprotein metabolism in the metabolic syndrome is well complemented by extensive cell biological data. Nutritional modifications and increased physical exercise may favourably alter lipoprotein transport in the metabolic syndrome by collectively decreasing the hepatic secretion of VLDL-apoB and the catabolism of HDL apoA-I, as well as by increasing the clearance of LDL-apoB. Pharmacological treatments, such as statins, fibrates or fish oils, can also correct the dyslipidaemia by several mechanisms of action including decreased secretion and increased catabolism of apoB, as well as increased secretion and decreased catabolism of apoA-I. The complementary mechanisms of action of lifestyle and drug therapies support the use of combination regimens to treat dyslipidaemia in the metabolic syndrome.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Tetrathiomolybdate therapy protects against concanavalin a and carbon tetrachloride hepatic damage in mice

Tetrathiomolybdate, an anticopper drug, has been shown to protect mice against pulmonary fibrosis from bleomycin. Our hypothesis is that it does so by inhibiting fibrosis-inducing cytokines. Indeed, we have good evidence, not yet published, that tetrathiomolybdate inhibits pulmonary levels of transforming growth factor-beta and tumor necrosis factor-alpha expression in these bleomycin experiments. Herein, we evaluate tetrathiomolybdate's effectiveness in mitigating hepatitis and fibrosis in mice from the hepatotoxins, concanavalin A and carbon tetrachloride, and its inhibition of cytokines as a possible mechanism. In short-term experiments, concanavalin A elevated serum amino leucine transferase levels several fold, and tetrathiomolybdate completely prevented this increase. In additional experiments, tetrathiomolybdate therapy reversed the elevated serum transaminase levels despite continued concanavalin A injections, with nearly significant serum interleukin-1beta inhibition. Concanavalin A given for 12 weeks produced mild fibrosis, whereas concomitant tetrathiomolybdate treatment resulted in normal histology. Carbon tetrachloride given for 12 weeks resulted in very high serum amino leucine transferase levels, high serum transforming growth factor-beta levels, cirrhosis as seen histologically, and increase in liver hydroxyproline, a measure of fibrosis. Concomitant tetrathiomolybdate partially and significantly protected against increases in amino leucine transferase and transforming growth factor-beta, fully protected against the increase in hydroxyproline, and resulted in normal histology. In conclusion, tetrathiomolybdate protects against the hepatitis and fibrosis produced by these hepatotoxins, probably by inhibiting the excessive increase in inflammatory and fibrotic cytokines.     

Using genetic markers to estimate the pollen dispersal curve

Pollen dispersal is a critical process that shapes genetic diversity in natural populations of plants. Estimating the pollen dispersal curve can provide insight into the evolutionary dynamics of populations and is essential background for making predictions about changes induced by perturbations. Specifically, we would like to know whether the dispersal curve is exponential, thin-tailed (decreasing faster than exponential), or fat-tailed (decreasing slower than the exponential). In the latter case, rare events of long-distance dispersal will be much more likely. Here we generalize the previously developed TWOGENER method, assuming that the pollen dispersal curve belongs to particular one- or two-parameter families of dispersal curves and estimating simultaneously the parameters of the dispersal curve and the effective density of reproducing individuals in the population. We tested this method on simulated data, using an exponential power distribution, under thin-tailed, exponential and fat-tailed conditions. We find that even if our estimates show some bias and large mean squared error (MSE), we are able to estimate correctly the general trend of the curve - thin-tailed or fat-tailed - and the effective density. Moreover, the mean distance of dispersal can be correctly estimated with low bias and MSE, even if another family of dispersal curve is used for the estimation. Finally, we consider three case studies based on forest tree species. We find that dispersal is fat-tailed in all cases, and that the effective density estimated by our model is below the measured density in two of the cases. This latter result may reflect the difficulty of estimating two parameters, or it may be a biological consequence of variance in reproductive success of males in the population. Both the simulated and empirical findings demonstrate the strong potential of TWOGENER for evaluating the shape of the dispersal curve and the effective density of the population (d(e)).     

Oncostatin M-stimulated apical plasma membrane biogenesis requires p27(Kip1)-regulated cell cycle dynamics

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.     

Polarized membrane traffic and cell polarity development is dependent on dihydroceramide synthase-regulated sphinganine turnover

Sphingoid bases have been implicated in various cellular processes including cell growth, apoptosis and cell differentiation. Here, we show that the regulated turnover of sphingoid bases is crucial for cell polarity development, i.e., the biogenesis of apical plasma membrane domains, in well-differentiated hepatic cells. Thus, inhibition of dihydroceramide synthase or sphinganine kinase activity with fumonisin B1 or N,N-dimethylsphingosine, respectively, dramatically perturbs cell polarity development, which is due to increased levels of sphinganine. Consistently, reduction of free sphinganine levels stimulates cell polarity development. Moreover, dihydroceramide synthase, the predominant enzyme responsible for sphinganine turnover, is a target for cell polarity stimulating cAMP/protein kinase A (PKA) signaling cascades. Indeed, electrospray ionization tandem mass spectrometry analyses revealed a significant reduction in sphinganine levels in cAMP/PKA-stimulated cells. These data suggest that sphinganine turnover is critical for and is actively regulated during HepG2 cell polarity development. Previously, we have identified an apical plasma membrane-directed trafficking pathway from the subapical compartment. This transport pathway, which is part of the basolateral-to-apical transcytotic itinerary, plays a crucial role in apical plasma membrane biogenesis. Here, we show that, as a part of the underlying mechanism, the inhibition of dihydroceramide synthase activity and ensuing increased sphinganine levels specifically perturb the activation of this particular pathway in the de novo apical membrane biogenesis.